An enantioselective and modular platform for C4'-modified nucleoside analogue synthesis enabled by intramolecular trans-acetalizations.

C4'-modified nucleoside analogues continue to attract global attention for their use in antiviral drug development and oligonucleotide-based therapeutics. However, current approaches to C4'-modified nucleoside analogues still involve lengthy (9-16 steps), non-modular routes that are unamenable to library synthesis. Towards addressing the challenges associated with their syntheses, we report a modular 5-step process to a diverse collection of C4'-modified nucleoside analogues through a sequence of intramolecular trans-acetalizations of readily assembled polyhydroxylated frameworks. Overall, the 2-3 fold reduction in step-count compares favorably to even recently reported biocatalytic approaches and should ultimately enable new opportunities in drug design around this popular chemotype.

Artificial intelligence assisted patient blood and urine droplet pattern analysis for non-invasive and accurate diagnosis of bladder cancer.

Bladder cancer is one of the most common cancer types in the urinary system. Yet, current bladder cancer diagnosis and follow-up techniques are time-consuming, expensive, and invasive. In the clinical practice, the gold standard for diagnosis remains invasive biopsy followed by histopathological analysis. In recent years, costly diagnostic tests involving the use of bladder cancer biomarkers have been developed, however these tests have high false-positive and false-negative rates limiting their reliability. Hence, there is an urgent need for the development of cost-effective, and non-invasive novel diagnosis methods. To address this gap, here we propose a quick, cheap, and reliable diagnostic method. Our approach relies on an artificial intelligence (AI) model to analyze droplet patterns of blood and urine samples obtained from patients and comparing them to cancer-free control subjects. The AI-assisted model in this study uses a deep neural network, a ResNet network, pre-trained on ImageNet datasets. Recognition and classification of complex patterns formed by dried urine or blood droplets under different conditions resulted in cancer diagnosis with a high specificity and sensitivity. Our approach can be systematically applied across droplets, enabling comparisons to reveal shared spatial behaviors and underlying morphological patterns. Our results support the fact that AI-based models have a great potential for non-invasive and accurate diagnosis of malignancies, including bladder cancer.

In silico analysis of the possible crosstalk between O-linked ß-GlcNAcylation and phosphorylation sites of Disabled 1 adaptor protein in vertebrates.

Disabled 1 (Dab1) is an adaptor protein with essential functions regulated by reelin signaling and affects many biological processes in the nervous system, including cell motility, adhesion, cortical development, maturation, and synaptic plasticity. Posttranslational modifications directly guide the fates of cytoplasmic proteins to complete their functions correctly. Reciprocal crosstalk between O-GlcNAcylation and phosphorylation is a dynamic modification in cytoplasmic proteins. It modulates the functions of the proteins by regulating their interactions with other molecules in response to the continuously changeable cell microenvironment. Although Dab1 contains conserved recognition sites for phosphorylation in their N-terminal protein interaction domain, the O-ß-GlcNAcylation and phosphorylation sites of human Dab1 sequence, their reciprocal crosstalk, and potential kinases catalyzing the phosphorylation remain unknown. In this study, we determined potential thirty-seven O-ß-GlcNAcylation and sixty-seven phosphorylation sites. Conserved twenty-one residues of these glycosylated sites were also phosphorylated with various kinases, including ATM, CKI, DNAPK, GSK3, PKC, PKG, RSK, cdc2, cdk5, and p38MAPK. In addition, we analyzed these conserved sites at our constructed two- and three-dimensional structures of human Dab1 protein. Dab1 protein models were frequently composed of coil structures as well as α-helix and ß-strands. Many of these conserved crosstalk sites between O-ß-GlcNAcylation and phosphorylation were localized at the coil region of the protein model. These findings may guide biochemical, genetic, and glyco-biology based on further experiments about the Dab1 signaling process. Understanding these modifications might change the point of view of the Dab1 signaling process and treatment for pathological conditions in neurodegenerative diseases such as Alzheimer's disease.

Exploring the Candidate Terminal Glycan Profile in Neural Regeneration of the Sea Urchin Paracentrotus lividus, Using Lectin Blotting and Mass Spectrometry.

Glycans are expressed as conjugates of glycoproteins, glycolipids, and proteoglycans. The huge diversity of glycans on glycoconjugates contributes to many biological processes, from glycan-based molecular recognition to developmental events, such as regeneration in the nervous system. Echinoderms, which have a close phylogenetic relationship with chordates, are an important group of marine invertebrates for body regeneration. Although many major roles of glycans on glycoconjugates are known, their role in the glycosylation profile of the nervous system in sea urchins is poorly understood. In this study, we aimed to determine the terminal glycan profile by lectin blotting and to quantify sialic acids by the capillary liquid chromatography electrospray ionization tandem mass spectrometry system in the nervous tissue of the sea urchin Paracentrotus lividus. We determined the N-acetyl-D-glucosamine, mannose, and sialic acids (mainly α2,3 linked) by lectin blotting and five types of sialic acids (N-glycolylneuraminic acid, N-acetylneuraminic acid, 9-O-acetyl-N-alycolylneuraminic acid, 5-N-acetyl-9-O-acetyl-N-acetylneuraminic acid, and di-O-acetylated-N-alycolylneuraminic acid) by capillary liquid chromatography electrospray ionization tandem mass spectrometry. This potential first description of the terminal glycan profile in the nervous system of the sea urchin is expected to help us understand its role in nervous system development and regeneration.

In silico analysis of glycosylation pattern in 5th-6th repeat sequence of reelin glycoprotein.

Reelin is an extracellular matrix glycoprotein that plays a key role in cortical development, maturation, synaptic plasticity, and memory formation in the adult mammalian brain. Glycosylation is a significant post- and co-translational modification of proteins. Although glycosylation contributes to the characteristic of proteins from their production to molecular interactions, the knowledge about the glycosylation pattern of reelin is very limited. In this study, we aimed to predict the potential glycosylation pattern of the 5th-6th repeat of central reelin fragment that responsible for their signaling, by using in silico methods. We found that the predicted glycosylation pattern of the 5th-6th repeat of human reelin was highly conserved between vertebrate species. However, this conservation was not observed in analyzed invertebrates. For the first time, we described the sites of glycosylation at a three-dimensional protein structure in human reelin. Because the sites were very closed to EGF-like repeats and receptor binding sites, they could contribute the interaction with a partner of reelin in addition to the effect of thermostability to protein. Many of the residues related glycosylation were also conserved in analyzed species. These findings may guide biochemical, genetic, and glycobiology base on further experiments about reelin glycosylation. The understanding of reelin glycosylation might change the point of view of treatment for many pathological conditions in neurodegenerative diseases such as Alzheimer's disease. Communicated by Ramaswamy H. Sarma.

Determination of terminal glycan and total monosaccharide profiles of reelin glycoprotein in SH-SY5Y neuroblastoma cell line by lectin blotting and capillary liquid chromatography electrospray ionization-ion trap tandem mass spectrometry system.

Reelin (400 kDa) is an extracellular matrix glycoprotein that is a key regulator of the many significant biological processes including the brain formation, cell aggregation, and dendrite formation. The glycosylation contributes to the nature of the protein through folding, localization and trafficking, solubility, antigenicity, biological activity, and half-life. Although reelin is to be known as a glycoprotein, the knowledge of its glycosylation is very limited. In this study, we aimed to characterize the terminal glycan profile of reelin by lectin blotting and monosaccharide analysis of glycan chains by capillary liquid chromatography electrospray ionization ion trap tandem mass spectrometry (CapLC-ESI-MS/MS) in SH-SY5Y neuroblastoma cell line. According to our results, reelin was detected in different protein fragments (310, 250, and 85 kDa) in addition to full-length form (400 kDa) in the cell line. The reelin glycoprotein was found to carry the ß-N-Acetylglucosamine, α-Mannose, ß-Galactose, and α-2,3 and α2,6 linked sialic acids by lectin blotting. Nevertheless, these terminal monosaccharides were found in different intensity according to reelin fragments. Besides, we purified a reelin fragment (250 kDa), and we analyzed it for their monosaccharide by CapLC-ESI-MS/MS. We found that reelin contained five types of monosaccharides, which were consisted of N-Acetylgalactosamine, N-Acetylglucosamine, Galactose, Glucose, Mannose and Sialic acid, from high to low abundance respectively. The present results provide a valuable guide for biochemical, genetic, and glycobiology based further experiments about reelin glycosylation in cancer perspective.